Method for the direct detection and/or quantification of at least one compound with a molecular weight of at least 200

ABSTRACT

The present invention relates to method for the direct detection and/or quantification of at least one compound with a molecular weight of at least 200, wherein the compound to be detected and/or quantified is a chemically complex molecule, wherein said chemically complex molecule is substituted with at least two groups —R, wherein each R group means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that at least two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2, wherein the compound or compounds to be detected and/or quantified are within a biological matrix, wherein said biological matrix is a biological fluid, a biological tissue, stomach contents, intestine contents, stool sample or a culture cells, wherein the method comprises performing a chromatography and identifying the retention time and/or the intensity of the signal by means of a mass or radioactivity detector.

The present invention relates to a direct liquid chromatographymethodology for the quantification of chemical complex molecules,especially wherein the chemically complex molecule is an inositolpolyphosphate, selected from the group containing from 2 to 6 phosphategroups.

BACKGROUND ART

The analytical determination of phosphorus containing chemically complexmolecules is fraught with serious difficulties due mainly to theirphysicochemical properties. These molecules are highly charged and dueto the presence of complex functional groups, such as phosphates orphosphonates, show a different ionization degree depending on pH. Thisissue becomes especially relevant for molecules with more than onephosphorus containing group.

Likewise, the absorption bands lack within the UV-Vis spectrum region inthe majority of these chemically complex molecules hinder itsquantification by direct spectrophotometric methods.

Furthermore, the determination of chemically complex molecules within abiological matrix involves additional difficulties due to the matrixeffect, so new bioanalytical methods for routine use must be developedand validated.

The low concentration of the analyte compared to other components in thematrix can suppress the analyte response. These effects can causedifferences in response between sample in matrix and standards, leadingto difficulties in quantitative analysis and compound identification(Biol Pharm Bull 25, 547-557; 2002).

For example, bisphosphonates present two phosphonic groups per moleculewhich lend a strongly ionic character and increased polarity.Additionally, the majority of the members of this family lacks ofchromophores excluding convenient direct UV detection (J Pharm BiomedAnal 48, 483-496; 2008). Other bioanalytical methods have been developedfor the determination of bisphosphonates, adding a derivatization stepfor its determination or fragmentation not being a direct method,understood direct method as the determination of the molecule per se oran adduct of this molecule, what provides a more sensible, specific,accurate, and robust method but specially more applicable, quick andreliable methodologies for routine analysis (J Chromatogr B 877,3159-3168; 2009, Int J Mass Spectrom 295, 85-93; 2010, J Mass Spectrom37, 197-208; 2002).

Other example of phosphorus containing chemically complex molecule arepyrophosphates. Pyrophosphates differ from the bisphosphonates in thecarbon atom that binds the two phosphorus atoms (Carbon atom of thebisphosphonates (P—C—P) is replaced by an oxygen (P—O—P)).

Nucleotides are also an example of chemically complex molecules, highlycharged and with polar nature (due to the presence of one or morephosphate groups), thus being this invention especially relevant forthose with more than one phosphate group.

The selective determination of inositol polyphosphates (from 2 to 6phosphate groups), together with other related impurities and especiallyin biological matrices is one of the major analytical breakthroughsfaced in this invention. In the extreme case, inositol hexaphosphate,also known as InsP6 or IP6, presents 12 dissociable protons, having pKavalues that range from negative values to more than 10 (Carbohydr Res46, 159-171; 1976).

Numerous analytical methods for InsP6 quantification have been describedin bibliography. Nevertheless, most of them have been developed fordetermination in simple matrices, (e.g. those that come from methods forfood extracts or pharmaceutical preparations), where concentrations ofInsP6 are higher than expected in biological matrices.

In the case of complex matrices (generally, those with biologicalorigin) previous cited methods have not sensitivity and specificityenough for InsP6 determination when concentrations to be quantified arelow, as the case of plasma, urine, other biological fluids, tissues orcells.

A high sensible and selective method is needed for the quantification ofinositol polyphosphates in this kind of matrices, and together withtheir special physicochemical properties, make most of the currentmethods useless.

Moreover, although some of the current methods could detect inositolpolyphosphates, they are not reproducible and solid enough for their usein clinical studies, when routine sample analysis must be performed.

The WO/2009/109647 discloses the determination of the amount of inositolphosphate (IP1 or InsP1, only 1 phosphate group) in the sample (urine,plasma) by using LC-ESI/MS/MS assay. The US20100136600 discloses thedetermination of myo-inositol in the sample (urine, plasma) by usingLC-MS/HPLC-MS techniques. However, neither of them disclose the IP6determination method and also the determination technique disclosed inthese patents may not be effective/suitable for IP2-IP6 determinationbecause IP2-IP6 are highly charged molecules as compared to the inositol(no phosphate groups, no charge) or IP1 (only one phosphate group,limited charge).

Indirect methods for the determination of InsP6 and InsP3 in plasma werepreviously developed by using gas chromatography-mass detection analysisof HPLC chromatographic fractions which involves hydrolysis of IP6 andfurther, determination of inositol or phosphate, leading to a highlytime consuming methodology (3 days per sample) and poorly accurateresults (Life Sci 71, 1535-1546; 2002).

Direct HPLC-MS for its quantification in plants extracts and in vitroculture cells have already been described (Mass Spectrom 23, 705-712;2009). However, the HPLC conditions described make the method uselessfor all the other biological matrices.

HPLC/MS with thermospray ionization allows the determination of inositolphosphates, but the lack of sensitivity is stated in the paper as alimitation for biological applications (Biomed Environ Mass Spectrom 19,597-600; 1990).

Other indirect methods for the determination of IP6 in human urine arebased on the total phosphorus measurement of purified extracts of phyticacid are described. In this case, a specific pretreatment of the sampleis required to avoid interference from other phosphorus containingcompounds accompanying phytic acid in urine such as phosphate orpyrophosphate (Anal Chem 75, 6374-6378; 2003, Anal Chim Acta 510, 41-43;2004). One of the drawbacks of these methods is that they are limitedto, due to sensitivity and selectivity issues, urine samples, not beingapplicable to tissues or blood samples and to the quantification ofrelated impurities. In addition, the indirect determination of IP6through total phosphorus is an extrapolation which leads to systematichigh values since all the quantified phosphorus is identified as IP6.

Another indirect method is described for the determination of phytate inhuman urine. The method is based on hydrolysis of the phytate anddetermination of myo-inositol, one of the hydrolysis products(Chromatographia 60, 265-268; 2004). This is a time-consumingmethodology (2 days per sample) due to the need of acidic hydrolysis.Furthermore, the limited sensitivity and the lack of specificity of thehydrolysis make the method useless for other biological matrices and forthe co-quantification of related impurities.

Other documents considered as prior art for IP6 determination inbiological samples (mainly in urine) are cited below:

March et al described in 2001 a unique methodology to quantify inositolphosphates in blood, but also applicable to urine and tissues (JChromatogr B 757, 247-255; 2001). It's a time-consuming indirectmethodology (3 days per sample) based on the enzymatic hydrolysis of IP6and determination of the inositol molecule (hydrolysate) through gaschromatography and mass spectrometry. It's a sensitive method, althoughindirect, based on different principles from the present invention,which in any case fails in the specificity, since all inositolphosphates are hydrolysed inespecifically and the whole hydrolysate isquantified as IP6.

Fluorescence has been widely used for the quantification of IP6,although the poor sensitivity just allows the application in foods andin urine as biological samples (Anal Chim Acta 605, 185-191; 2007).

They are totally different methods from the invention here described,which in any case fail to determine related impurities and are notapplicable to other biological matrices different from urine.

SUMMARY OF THE INVENTION

The present invention relates to a direct liquid chromatographymethodology for the quantification of chemical complex molecules,understood under the scope of this invention as molecules with least twophosphorus containing groups and a molecular weight of at least 200.These complex molecules are highly polar and highly charged due to thepresence of several ionizable groups. In addition, in many cases thelack of absorption bands in the UV-visible spectrum region make thesemolecules invisible through the classical techniques.

The identification and quantification of the target compound isperformed through direct detection. Under the scope of the presentinvention, direct detection is defined as theidentification/quantification of the analyte by means of a property ofthe compound (or its ions or salts thereof) ‘as is’ or a property of thecompound forming an adduct (such as but not limited to ionicassociates), metabolites, preferably phase II metabolites, of the parentcompound, including fragments of the main analyte in mass detection.This property measured by direct detection can be, but is not limitedto, mass detection, conductivity, radioactivity (thus the analyte can beradiolabelled), NMR, being the mass detection a preferred one. Thepresent inventors have found that, when the mass is the chemical complexmolecule is lower than 200, selectivity is compromised at those levelsdue to the interference of several compounds from the mobile phases andthe atmosphere.

In addition, the sensitivity of the present invention can be around 1pmol, which is in the high range of the currently availablemethodologies. Moreover, the combination of the sensitivity with thespecificity/selectivity of the present invention allows its applicationin all kind of biological matrices as well as the quantification of themain analyte together with related impurities in the case of qualitycontrol of active pharmaceutical ingredients (s), medical foods,reagents, food additives, pharmaceutical compositions or nutraceuticals.

“Biological matrix” refers to an environment that may or may not beisolated from a warm-blooded animal. Non-limiting examples of biologicalmatrices are: fluid, tissue, stomach contents, intestine contents, stoolsample, culture cells, urine, feces, blood, serum, plasma, saliva,perspiration, tissue fluid, cellular cytoplasm, hepatocytes, microsomes,S9 fractions, tissues, such as muscle tissue, hepatic tissue, cardiactissue, renal tissue and other bodily environments and/or matrices of awarm-blooded animal, preferably a human. A biological matrix may bepresent in solution or in solid form or a mixture thereof and may bepresent in or as part of a living organism or may be isolated from aliving organism such that it forms a sample therefrom. The methodsherein disclosed provide excellent results even working with abiological matrix comprising very low levels of chemically complexmolecule, being preferred concentrations those below 0.001 μmol/mg (forsolid matrices) or μmol/μl (for matrices in other physical states), evenmore preferred below 0.000001 μmol/mg or μmol/μl, and even morepreferred amounts in the range of 0.0000001 μmol/mg or μg/μl. Thebiological matrix may be biological fluids, which can be, but are notlimited to, blood, plasma, serum, urine, saliva, lymphatic liquid,cerebrospinal fluid and mixtures thereof, preferably blood or plasma.The biological matrix may be also a biological tissue, which can be, butis not limited to lung, liver, kidney, heart, blood vessels, brain,bone, skin, muscle, nervous tissue, vascular tissue and mixturesthereof, preferably heart tissue.

The method as herein disclosed, it is also a reliable methodology forbiological matrices of different species, for example, but not limitedto, rats, mice, dogs, monkeys, humans, pigs, minipigs, rabbits, guineapigs.

Various aspects of the present invention are described below.

Therefore, the method as herein disclosed provides a method for thedirect detection and/or quantification of at least one compound with amolecular weight of at least 200, wherein the compound to be detectedand/or quantified is a chemically complex molecule, wherein saidchemically complex molecule is substituted with at least two groups —R,preferably a C3-C7 cycloalkyl, wherein said C3-C7 cycloalkyl issubstituted with at least two groups —R, wherein each R group meansindependently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that atleast two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2,including ions or salts thereof, wherein the compound or compounds to bedetected and/or quantified are within a biological matrix, wherein saidbiological matrix is a biological fluid, a biological tissue, stomachcontents, intestine contents, stool sample or a culture cells, whereinthe method comprises performing a chromatography and identifying theretention time and/or the intensity of the signal by means of a mass orradioactivity detector.

According to the first aspect of the present invention there is provideda method for the direct detection and/or quantification of at least onecompound with a molecular weight of at least 200, wherein the compoundto be detected and/or quantified is a chemically complex molecule,wherein said chemically complex molecule is substituted with at leasttwo groups —R, preferably a C3-C7 cycloalkyl, wherein said C3-C7cycloalkyl is substituted with at least two groups —R, wherein each Rgroup means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with theproviso that at least two R are independently selected from —P(O)(OH)2and —OP(O)(OH)2, including ions or salts thereof, wherein the compoundor compounds to be detected and/or quantified are within a biologicalmatrix, wherein said biological matrix is a biological fluid, abiological tissue, stomach contents, intestine contents, stool sample ora culture cells, wherein the method for the direct detection and/orquantification comprises at least the following steps:

i) preparing at least one standard sample, preferably of a knownconcentration or from a known concentration, of the chemically complexmolecule to be detected and/or quantified;

ii) introducing the standard sample into the stream of a solvent system;wherein the solvent system is a polar solvent or a solvent mixturecomprising at least one polar solvent; wherein the pH of at least onesolvent which forms the solvent system has been preferably buffered,between 7 and 14.iii) passing the sample and the solvent system through at least onechromatographic column wherein the column is essentially filled ofsmalls particles of a stationary phase, preferably a non-polarstationary phase, while maintaining the pressure of the system between 5and 1500 atm;iv) identifying the retention time and/or quantifying the intensity ofthe signal of when the chemically complex molecule is eluated, by meansof a mass or radioactivity detector;v) preparing a sample of the chemically complex molecule from abiological matrix comprising the chemically complex molecule to bedetected and/or quantified, wherein the process to prepare the samplecomprises at least dissolving totally or partially the biological matrixto form a solution or a slurry, and, if necessary treating the solutionor the slurry to remove particles in suspension;vi) repeating the steps (ii) and (iii) with the sample prepared in thestep (v); together or sequentially with the standardvii) detecting and/or quantifying the presence of the chemically complexmolecule by comparison of retention time and/or intensity of signal ofthe standard sample or the standard samples, or by comparison ofretention time and/or intensity of signal obtained from previous studiesor from literature.

According to the second aspect of the present invention there isprovided a method for analysing an API, a medical food, a reagent, afood additive, a pharmaceutical composition or nutraceutical, whereinthe API, the medical food, the reagent, the food additive, thepharmaceutical composition or the nutraceutical comprise at least onecompound with a molecular weight of at least 200, wherein said compoundis a chemically complex molecule, wherein said chemically complexmolecule is substituted with at least two groups —R, preferably a C3-C7cycloalkyl, wherein said C3-C7 cycloalkyl is substituted with at leasttwo groups —R, wherein each R group means independently —OH, —OP(O)(OH)2or —P(O)(OH)2, with the proviso that at least two R are independentlyselected from —P(O)(OH)2 and —OP(O)(OH)2, including ions or saltsthereof, wherein this compound can also be quantified together with itsrelated impurities, wherein the method for analysing comprises at leastthe following steps:

i) preparing at least one standard sample, preferably of a knownconcentration or from a known concentration of the chemically complexmolecule to be detected and/or quantified;

ii) introducing the standard sample into the stream of a solvent system;wherein the solvent system is a polar solvent or a solvent mixturecomprising at least one polar solvent; wherein the pH of at least onesolvent which forms the solvent system has been preferably buffered,between 7 and 14.iii) passing the sample and the solvent system through at least onechromatographic column wherein the column is essentially filled ofsmalls particles of a stationary phase, preferably a non-polarstationary phase, while maintaining the pressure of the system between 5and 1500 atm;iv) identifying the retention time and/or quantifying the intensity ofthe signal of when the chemically complex molecule is eluated, by meansof a detector capable to detect the eluation of the chemically complexmolecule;v) preparing a sample of the chemically complex molecule from the API, amedical food, a reagent, a food additive, a pharmaceutical compositionor nutraceutical comprising the chemically complex molecule to bedetected and/or quantified, wherein the process to prepare the samplecomprises at least dissolving totally or partially the API, medicalfood, reagent, food additive, pharmaceutical composition ornutraceutical to form a solution or a slurry, and, if necessary,treating the solution or the slurry to remove particles in suspension;vi) repeating the steps (ii) and (iii) with the sample prepared in thestep (v); together or sequentially with the standard.vii) detecting and/or quantifying the presence of the chemically complexmolecule by comparison of retention time and/or intensity of signal ofthe standard sample or the standard samples, or by comparison ofretention time and/or intensity of signal obtained from previous studiesor from literature.

According to the third aspect of the present invention there is providea process for preparing a drug, a medical food, a pharmaceuticalcomposition or a nutraceutical pharmaceutical composition comprising atleast one compound with a molecular weight of at least 200, wherein saidcompound is a chemically complex molecule, wherein said chemicallycomplex molecule is substituted with at least two groups —R, preferablya C3-C7 cycloalkyl, wherein said C3-C7 cycloalkyl is substituted with atleast two groups —R, wherein each R group means independently —OH,—OP(O)(OH)2 or —P(O)(OH)2, with the proviso that at least two R areindependently selected from —P(O)(OH)2 and —OP(O)(OH)2, including ionsor salts thereof, wherein the drug, a medical food, a pharmaceuticalcomposition or a nutraceutical has to have a predetermined percentage ofthe chemically complex molecule, wherein the process comprises:obtaining a batch of a drug, a medical food, a pharmaceuticalcomposition or a nutraceutical pharmaceutical; measuring the percentageof purity the chemically complex molecule of the batch by a processcomprising the method according to the second aspect; and including thedrug, the medical food, the pharmaceutical composition or thenutraceutical batch only if its percentage of the chemical complexmolecule so measured is within the requirements or predefinedspecifications, being the normal assay, referred to dry content, majorthan 60%, preferably major than 70% and more preferably major than 80%.

According to the fourth aspect of the present invention there isprovided a process for the production of a pharmaceutical compositioncomprising at least one compound with a molecular weight of at least200, wherein said compound is a chemically complex molecule, whereinsaid chemically complex molecule is substituted with at least two groups—R, preferably a C3-C7 cycloalkyl, wherein said C3-C7 cycloalkyl issubstituted with at least two groups —R, wherein each R group meansindependently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that atleast two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2,including ions or salts thereof, wherein the process comprisesessentially the same process steps of a process for the production of afirst batch, provided that after analyzing by any of the method of thefirst aspect a biological isolated sample, preferably a blood or a serumsample, taken from subjects to whom the pharmaceutical compositionsobtained by the first batch were administrated, the calculated AUC andCmax are within the desire requirements and/or are bioequivalent to areference drug, medical food, pharmaceutical composition ornutraceutical. “AUC” refers to the area under a curve representing theconcentration of a compound or metabolite thereof in a biological fluid,e.g., plasma and blood, in a patient as a function of time followingadministration of the compound to the patient. Cmax is the maximumconcentration of a drug in the plasma or blood of a patient followingadministration of a dose of the drug or form of drug to the patient.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the present invention are described below.

The chemically complex molecule may be a bisphosphonate,hexametaphosphate or a C3-C7 cycloalkyl, wherein said C3-C7 cycloalkylis substituted with at least two groups —R, with the proviso that atleast two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2.The term “C3-C7 cycloalkyl” refers to saturated cyclic alkyl grouphaving from three to seven carbon atoms and includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Preferredchemically complex molecule is a C6 cycloalkyl. The chemically complexmolecule may be also a cycloalkyl derivative. Excellent results havebeen achieved when the chemically complex molecule is an inositolpolyphosphate.

The chemically complex molecule may be radiolabelled.

The standard sample prepared in step (ii) may be a reference standard,external standard, internal standard or standard addition, meaning thislatter a standard prepared by the standard addition method, alsodescribed below.

As used herein, the term “reference standard” refers to a compound thatmay be used both for quantitative and qualitative analysis of ananalyte, which could be, but not limited to, an active pharmaceuticalingredient. For example, the retention time of the compound in liquidchromatography (LC), for example HPLC or UPLC, allows for setting arelative retention time, thus making qualitative analysis possible. Theconcentration of the compound in solution before injection into an LCcolumn allows for comparison of the areas under the peaks in an LCchromatogram, thus making quantitative analysis possible.

As used herein, the term external standard refers to a chemical complexmolecule of known concentration. If used for determining concentration,external standards may be preferably added to the test sample at a knownconcentration, technique known as standard addition.

As used herein, the term “internal standard” refers to what is added toand subsequently detected and quantified in the sample. The addition ofthe internal standard can be before, during or after sample collectionor processing. The internal standard, as contemplated by the presentinvention, is a compound that is added to the sample, and this similarcompound is then quantified using the methods described herein.

The solvent system preferably comprises a polar organic solvent. Solventsystem comprises a water-miscible polar organic solvent are ofparticular interest. Preferred solvent systems comprise acetonitrile,methanol or mixtures thereof. More preferably the solvent systemcomprises acetonitrile.

Additionally the solvent system may comprise water. One of the mostpreferred solvent systems comprises, preferably consisting essentiallyof, water and acetonitrile.

The solvent system may be isocratic or gradient. In one preferredembodiment during the step (iii) the composition of the solvent systemis gradually increased, i.e. gradient, i.e. the strength of the mobilephase is gradually increased. Preferably the system starts with a highwater content at the moment when the sample and the solvent system startpassing throughout the column and then the water content is graduallydecreased during the elution of the chemically complex molecule.

Optionally, during the step (iii) the composition of the solvent systemand consequently the strength of the mobile phase is maintainedessentially unaltered.

In one particular embodiment, during the step (iii) the solvent systemcomprises at least one solvent, wherein the pH of such solvent has beenbuffered from 7 to 14, preferably the pH of such solvent has beenadjusted from 8 to 13, and more preferably from 8.5 to 12.

There are several techniques available to the skilled person to adjustthe pH. The pH could be modified with a substance capable of modifyingthe pH, typically, but not limited to, an acid, a base or a salt withacidic or basic properties. Amongst other, substances capable ofmodifying the pH can be an amine, an inorganic base, a salt of anorganic acid, or any mixtures thereof. Amine and, particularlytriethylamine, are preferred. The inorganic base may be a hydroxide,preferably KOH. It could be used as a salt of an organic acid, e.g.acetate.

In a particular embodiment, before injecting the already prepared sampleto the corresponding equipment, there is an additional pH control.Preferably, the pH is adjusted to the same pH as the mobile phase. Itcan be adjusted with an acid, a base or a salt, preferably an aminebase, and more preferably the amine is triethylamine.

As used herein, the term “chromatographic column” refers to a tubepacked with adsorbent particles and used to perform chromatography.Particularly interesting are column wherein the smalls particles of anon-polar stationary phase are silica based, preferably silica gelbased. Good results are obtained when the smalls particles are anon-polar stationary phase, preferably are silica gel based covalentlybonded to an alkyl chain group. The alkyl chain group may be a C₂-C₂₀alkyl, preferably a C₈-C₁₈ alkyl.

The small particles of a non-polar stationary phase may have a meanparticle size between 0.5 and 20 microns, preferably between 1.5 and 15,even more preferably between 5 and 10.

The pore size may range between 10 and 1000 Angstroms, preferablybetween 60 and 500 Angstroms, and even more preferably between 100 and300 Angstroms.

The reproducibility of the system and other advantages are improved whenthe temperature of the column during the step (iii) is maintainedessentially unaltered. Typically the temperature of the column duringthe step (iii) is maintained essentially unaltered between 10 and 70° C.Preferably, it is maintained essentially unaltered between 45 and 55°C., and more preferably is maintained essentially unaltered about 50° C.

The term “detector” refers to any device, apparatus, machine, component,or system that can detect the chemical complex molecule. Detectors mayor may not include hardware and software. In a mass detector (massspectrometer) the common detector includes and/or is coupled to a massanalyzer. Examples of detectors capable to detect the eluation of thechemically complex molecule is a mass spectrometers; a tandem massspectrometer (or triple quadrupole); a single quadrupole. These massdetectors can work with several operating modes, amongst others selectedion monitoring (SIM), multiple reaction monitoring (MRM), selectedreaction monitoring (SRM) and SCAN (negative/positive); or several ofthem. A radioactivity detector can also be used. For the second aspect,it is also possible to use a conductimetry, photometric and/or a NMRdetector.

The use of mass detector typically implies a further step, namely aprevious step of phase change and ionization after the chemicallycomplex molecule between the elution and the detection. The phase changeand ionization may be, but not limited to, a chemical ionization (APCI),atmospheric pressure photoionization (APPI) or electrospray ionization(ESI) or thermospray. The electrospray ionization (ESI) may be carriedout under N₂ at a temperature between 250 and 1000° C., preferablybetween 300 an 800° C., even more preferably between 400 and 600° C.

For pretreating the biological sample it may be necessary in the step(v) the use of a protein precipitating agent, preferably trichloroaceticacid for the pretreatment of the sample. This additional treatmentallows the simplification of the biological matrix, obtaining betterselectivity and increasing sensitivity, by reducing matrix effects. Step(v) may also comprise the use of a chelating agent for the pretreatmentof the sample, such as EDTA. These chelating agents reduce the freecalcium concentration in the biological sample, thus avoiding theformation of labile compounds together with the analyte, which wouldreduce the recuperation and compromise the accuracy of the methodology.

The biological matrix may be also preconcentrated, thus increasing thesensitivity of the method.

In another particular embodiment there is provided a method for thedirect detection and/or quantification of an inositol polyphosphatecontaining at least 2 phosphorus groups, or its ions or salts thereof,wherein the inositol polyphosphate is within a biological matrix,wherein said biological matrix is a biological fluid, a biologicaltissue, stomach contents, intestine contents, stool sample or a culturecells, wherein the method for the direct detection and/or quantificationcomprises at least the following steps:

i) preparing at least one standard sample, preferably of a knownconcentration or from a known concentration, of inositol polyphosphatecontaining at least 2 phosphorus groups to be detected and/orquantified;

ii) introducing the standard sample into the stream of a solvent system;wherein the solvent system consists of an aqueous solution of an amine;wherein the pH of the solvent has been buffered, between 8 and 13.

iii) passing the sample and the solvent system through at least onecolumn wherein the column is a reversed-phase stationary phase, beingthe particle size between 1.5-15 microns and the pore size between60-500 Angstroms.

iv) identifying the retention time and/or quantifying the intensity ofthe signal of when the inositol polyphoshate is eluated, by means of amass detector;

v) preparing a sample of the inositol polyphoshate from a biologicalmatrix comprising the inositol polyphoshate to be detected and/orquantified, wherein the process to prepare the sample comprises at leastdissolving totally or partially the biological matrix to form a solutionor a slurry, and, if necessary treating the solution or the slurry toremove particles in suspension;vi) repeating the steps (ii) and (iii) with the sample prepared in thestep (v); together or sequentially with the standard.vii) detecting and/or quantifying the presence of the inositolpolyphoshate by comparison of retention time and/or intensity of signalof the standard sample or the standard samples.

During the sample pretreatment, a step for solid-liquid separation maybe included, typically centrifugation or filtration, to remove any solidparticles that could have appeared during the process. In this step thetemperature is preferably controlled to 2-8° C. during the samplepretreatment, more preferably about 4° C.

The method as disclosed herein is useful for the analysis of chemicalcomplex molecules. One particular use of the methods as disclosed hereinis analysis of biologically matrix of human beings when the human beingis under a bioequivalence study. Another particular use of the methodsas disclosed herein is to simultaneously determine related impurities ofthe chemical complex molecule, especially of IP6.

The method of the present invention may be combined with otheranalytical techniques (RMN/Karl Fischer/GC/ICP/TG).

The term “metabolic profile” comprises the identification,semi-quantification and/or quantification of one or more metabolitesderived from a parent compound, in a biological matrix, which mainlyconsist of phosphorylation-dephosphorilation metabolic pathways.

The term “impurities profile” comprises the identification,semi-quantification and/or quantification of one or more impuritiesderived from a parent compound, mainly dephosphorylation relatedimpurities, in a formulation of an API, a medical food, a reagent, afood additive, a pharmaceutical composition or nutraceutical.

Further aspects/embodiments of the present invention can be found in thefollowing clauses:

Clause 1.—Method for the direct detection and/or quantification of atleast one compound with a molecular weight of at least 200, wherein thecompound to be detected and/or quantified is a chemically complexmolecule, wherein said chemically complex molecule is substituted withat least two groups —R, preferably a C3-C7 cycloalkyl, wherein saidC3-C7 cycloalkyl is substituted with at least two groups —R, whereineach R group means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, withthe proviso that at least two R are independently selected from—P(O)(OH)2 and —OP(O)(OH)2, including ions or salts thereof, wherein thecompound or compounds to be detected and/or quantified are within abiological matrix, wherein said biological matrix is a biological fluid,a biological tissue, stomach contents, intestine contents, stool sampleor a culture cells, wherein the method for the direct detection and/orquantification comprises at least the following steps:

i) preparing at least one standard sample, preferably of a knownconcentration or from a known concentration, of the chemically complexmolecule to be detected and/or quantified;

ii) introducing the standard sample into the stream of a solvent system;wherein the solvent system is a polar solvent or a solvent mixturecomprising at least one polar solvent; wherein the pH of at least onesolvent which forms the solvent system has been preferably buffered,between 7 and 14.iii) passing the sample and the solvent system through at least onecolumn wherein the column is essentially filled of smalls particles of astationary phase, preferably a non-polar stationary phase, whilemaintaining the pressure of the system between 5 and 1500 atm;iv) indentifying the retention time and/or quantifying the intensity ofthe signal of when the chemically complex molecule is eluated, by meansof a mass or radioactivity detector;v) preparing a sample of the chemically complex molecule from abiological matrix comprising the chemically complex molecule to bedetected and/or quantified, wherein the process to prepare the samplecomprises at least dissolving totally or partially the biological matrixto form a solution or a slurry, and, if necessary treating the solutionor the slurry to remove particles in suspension;vi) repeating the steps (ii) and (iii) with the sample prepared in thestep (v); together or sequentially with the standard.vii) detecting and/or quantifying the presence of the chemically complexmolecule by comparison of retention time and/or intensity of signal ofthe standard sample or the standard samples, or by comparison ofretention time and/or intensity of signal obtained from previous studiesor from literature.

Clause 2.—The method according to the preceding clause, wherein thechemically complex molecule is a bisphosphonate, hexametaphosphate,nucleotide or a C3-C7 cycloalkyl, wherein said C3-C7 cycloalkyl issubstituted with at least two groups —R.

Clause 3.—The method according to any one of the preceding clauses,wherein the chemically complex molecule is an inositol polyphosphate,selected from the group containing from 2 to 6 phosphate groups.

Clause 4.—The method according to any one of the preceding clauses,wherein the standard sample prepared in step (ii) is a referencestandard, external standard, internal standard or standard addition

Clause 5.—The method according to any one of the preceding clauses,wherein the solvent system comprises a water-miscible polar organicsolvent.

Clause 6.—The method according to any one of the preceding clauses,wherein during the step (iii) the composition of the solvent system isgradually increased, i.e. the strength of the mobile phase is graduallyincreased.

Clause 7.—The method according to any one of the clauses 1 to 5, whereinduring the step (iii) the composition of the solvent system is thestrength of the mobile phase is maintained essentially unaltered.

Clause 8.—The method according to any one of the preceding clauses,wherein the solvent system comprises water, and the pH of the water hasbeen adjusted from 7 to 14, preferably from 8 to 13, and more preferablyfrom 8.5 to 12 with a substance capable of modifying the pH, wherein thesubstance capable of modifying the pH is preferably an amine, aninorganic base, a salt of an organic acid, or any mixtures thereof, morepreferably an amine and/or NH₃, a salt of an organic acid or anhydroxide, and even more preferably triethylamine and/or NH₃.

Clause 9.—The method according to any one of the preceding clauses,further comprising at least 1 additional step of pH control, preferablyduring the pretreatment of the sample before injecting into theequipment.

Clause 10.—The method according to any one of the preceding clauses,wherein the smalls particles of a non-polar stationary phase are silicagel based covalently bonded to an alkyl chain group, wherein alkyl chaingroup is preferably a C₂-C₂₀ alkyl, and more preferably a C₈-C₁₈ alkyl.

Clause 11.—The method according to any one of the preceding clauses,wherein the detector capable to detect the eluation of the chemicallycomplex molecule is a mass spectrometer: a tandem mass spectrometer (ortriple quadrupole); a single quadrupole; working under any of thefollowing operating modes: selected ion monitoring (SIM); multiplereaction monitoring (MRM); selected reaction monitoring (SRM) and SCAN(negative/positive); or a combination of them.

Clause 12.—The method according to the preceding clause, furthercomprising a previous step of phase change and ionization of thechemically complex molecule between the elution and the detection.

Clause 13.—The method according to the preceding clause, wherein thephase change and ionization is chemical ionization (APCI), atmosphericpressure photoionization (APPI), electrospray ionization (ESI) orthermospray.

Clause 14.—The method according to the preceding clause, wherein theelectrospray ionization (ESI) is carried out under N₂ at a temperaturebetween 250 and 1000° C., preferably between 300 an 800° C., even morepreferably between 400 and 600° C.

Clause 15.—The method according to any one of the preceding clauses,wherein the step (v) also comprises the use of a protein precipitatingagent, preferably trichloroacetic acid for the pretreatment of thesample.

Clause 16.—The method according to any one of the preceding clauses,wherein the step (v) also comprises the use of a chelating agent for thepretreatment of the sample, preferably EDTA or its salts or any mixturethereof.

Clause 17.—The method according to any of the preceding clauses, whereinthe biological matrix is preconcentrated.

Clause 18.—The method according to any of the preceding clauses furthercomprising a step for solid-liquid separation during the pretreatment ofthe sample.

Clause 19.—The method according to any one of the preceding clauses,wherein the biological matrix is a biological fluid, preferably selectedfrom the list consisting of blood, plasma, serum, urine, saliva,lymphatic liquid, cerebrospinal fluid and mixtures thereof, preferablyblood and plasma.

Clause 20.—The method according to clauses 1 to 18, wherein thebiological matrix is a biological tissue, preferably selected from thelist consisting of lung, kidney, heart, brain, liver, blood vessels,bone, skin, muscle, nervous tissue, vascular tissue and mixturesthereof, preferably heart tissue.

Clause 21.—The method according to any one of the preceding clauses,wherein the biologically matrix to be analysed is an isolated sample ofa human being who is under an bioequivalence study.

Clause 23.—The method according to any of the preceding clauses, whereina surrogate matrix is used to prepare the calibration curve, preferablythe matrix used is formed by bovine serum albumin.

Clause 24.—The method according to any of the preceding clauses, when itis used to detect and quantify any of the metabolites of a compound in ametabolic profile.

Clause 25.—The method according to any one of the preceding clauses,wherein the compound is radiolabelled.

Clause 26.—Method for analysing an API, a medical food, a reagent, afood additive, a pharmaceutical composition or nutraceutical, whereinthe API, the medical food, the pharmaceutical composition or thenutraceutical comprise at least one compound with a molecular weight ofat least 200, wherein said compound is a chemically complex molecule,wherein said chemically complex molecule is substituted with at leasttwo groups —R, preferably a C3-C7 cycloalkyl, wherein said C3-C7cycloalkyl is substituted with at least two groups —R, wherein each Rgroup means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with theproviso that at least two R are independently selected from —P(O)(OH)2and —OP(O)(OH)2, including ions or salts thereof, this compound can alsobe quantified together with its related impurities, wherein the methodfor analysing comprises at least the following steps:

i) preparing at least one standard sample, preferably of a knownconcentration or from a known concentration of the chemically complexmolecule to be detected and/or quantified;

ii) introducing the standard sample into the stream of a solvent system;wherein the solvent system is a polar solvent or a solvent mixturecomprising at least one polar solvent; wherein the pH of at least onesolvent which forms the solvent system has been preferably buffered.iii) passing the sample and the solvent system through at least onecolumn wherein the column is essentially filled of smalls particles of astationary phase, preferably a non-polar stationary phase, whilemaintaining the pressure of the system between 5 and 1500 atm;iv) indentifying the retention time and/or quantifying the intensity ofthe signal of when the chemically complex molecule is eluated, by meansof a detector capable to detect the eluation of the chemically complexmolecule;v) preparing a sample of the chemically complex molecule from the API, amedical food, a reagent, a food additive, a pharmaceutical compositionor nutraceutical comprising the chemically complex molecule to bedetected and/or quantified, wherein the process to prepare the samplecomprises at least dissolving totally or partially the API, apharmaceutical composition or nutraceutical to form a solution or aslurry, and, if necessary, treating the solution or the slurry to removeparticles in suspension;vi) repeating the steps (ii) and (iii) with the sample prepared in thestep (v); together or sequentially with the standard.vii) detecting and/or quantifying the presence of the chemically complexmolecule by comparison of retention time and/or intensity of signal ofthe standard sample or the standard samples.

Clause 27.—The method according to the preceding clause, when it is usedto detect and quantify any of the impurities of a compound in animpurities profile.

Clause 28.—A process for preparing a drug, a medical food, a reagent, afood additive, a pharmaceutical composition or a nutraceuticalpharmaceutical composition comprising at least one compound with amolecular weight of at least 200, wherein said compound is a chemicallycomplex molecule, wherein said chemically complex molecule issubstituted with at least two groups —R, preferably a C3-C7 cycloalkyl,wherein said C3-C7 cycloalkyl is substituted with at least two groups—R, wherein each R group means independently —OH, —OP(O)(OH)2 or—P(O)(OH)2, with the proviso that at least two R are independentlyselected from —P(O)(OH)2 and —OP(O)(OH)2, including ions or saltsthereof, wherein the drug, a medical food, a pharmaceutical compositionor a nutraceutical has to have a predetermined percentage of thechemically complex molecule, wherein the process comprises: obtaining abatch of a drug, a medical food, a pharmaceutical composition or anutraceutical pharmaceutical; measuring the percentage of purity thechemically complex molecule of the batch by a process comprising themethod according to the preceding clause; and including the drug, themedical food, the pharmaceutical composition or the nutraceutical batchonly if its percentage of the chemical complex molecule so measured iswithin the requirements or specifications, preferably major than 70% byweight, and more preferably major than 80% by weight (referred to drycontent)

Clause 29.—A process for the production of a pharmaceutical compositioncomprising at least one compound with a molecular weight of at least200, wherein said compound is a chemically complex molecule, whereinsaid chemically complex molecule is substituted with at least two groups—R, preferably a C3-C7 cycloalkyl, wherein said C3-C7 cycloalkyl issubstituted with at least two groups —R, wherein each R group meansindependently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that atleast two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2,including ions or salts thereof, wherein the process comprisesessentially the same process steps of a process for the production of afirst batch, provided that after analysing a biological isolated sampleby any of the method of clauses 1 to 22, preferably a blood or a serumsample, taken from subjects to whom the pharmaceutical compositionsobtained by the first batch were administrated, the calculated AUC andCmax are within the desire requirements and/or are bioequivalent to areference drug.

Clause 30.—Metabolic profile of a compound with a molecular weight of atleast 200, wherein the compound to be detected and/or quantified is achemically complex molecule, wherein said chemically complex molecule issubstituted with at least two groups —R, preferably a C3-C7 cycloalkyl,wherein said C3-C7 cycloalkyl is substituted with at least two groups—R, wherein each R group means independently —OH, —OP(O)(OH)2 or—P(O)(OH)2, with the proviso that at least two R are independentlyselected from —P(O)(OH)2 and —OP(O)(OH)2, including ions or saltsthereof, wherein the compound or compounds are within a biologicalmatrix, wherein said biological matrix is a biological fluid, abiological tissue, stomach contents, intestine contents, stool sample ora culture cells.

Clause 31.—Impurities profile of a compound with a molecular weight ofat least 200, wherein the compound to be detected and/or quantified is achemically complex molecule, wherein said chemically complex molecule issubstituted with at least two groups —R, preferably a C3-C7 cycloalkyl,wherein said C3-C7 cycloalkyl is substituted with at least two groups—R, wherein each R group means independently —OH, —OP(O)(OH)2 or—P(O)(OH)2, with the proviso that at least two R are independentlyselected from —P(O)(OH)2 and —OP(O)(OH)2, including ions or saltsthereof, wherein the compound or compounds are present in a formulationof an an API, a medical food, a reagent, a food additive, apharmaceutical composition or nutraceutical

DESCRIPTION OF THE FIGURES

FIG. 1. UPLC®-MS chromatogram obtained for IP6 in rat plasma aftergradient-elution reversed-phase chromatography of example 1.

FIG. 2. HPLC-MS chromatogram obtained for IP6 in rat urine aftergradient-elution reversed-phase chromatography of example 4.

FIG. 3. Typical Chromatogram of an API sample (detail in the case of thesample) of example 8 (impurities profile). FIG. 3a : Blank. FIG. 3b :Sample (P: Phosphate, IP6: inositol hexaphosphate; 1, 2, 3 and 4:Related impurities).

FIG. 4. Typical Chromatogram of ATP for a plasma rat sample (Example 9).

FIG. 5. Typical Chromatogram of IP3 for a plasma rat sample (Example10).

FIG. 6. Typical Chromatogram of IP6 in human urine (Example 11).

FIG. 7. Typical Chromatogram of IP6 using a solution of 30 mg BSA(Bovine Serum Albumin)/mL in PBS as a surrogate matrix for plasma orserum (Example 12).

FIG. 8. Typical Chromatogram of the impurities present in a Phytic Acidsolution: IP5, IP4, IP3 and m/z 779 (Example 13)

FIG. 9. Typical Chromatogram of Phytic Acid metabolites m/z 779, m/z 740y m/z 579 after incubation of Phytic Acid in rat hepatocytes (Example14)

FIG. 10. Typical Chromatogram of Phytic Acid metabolites m/z 499, m/z419, m/z 339 y m/z 259 after incubation of Phytic Acid in rathepatocytes (Example 14)

FIG. 11. Typical Chromatogram of Phytic Acid after incubation of PhyticAcid in rat hepatocytes (Example 14)

FIG. 12. Typical Chromatogram of IP5 in rat plasma (Example 15)

FIG. 13. Typical Chromatogram of Phytic Acid metabolites (metabolicprofile) in rat plasma (Example 16)

FIG. 14. Typical Chromatogram of Phytic Acid metabolites (metabolicprofile) in dog plasma (Example 17)

FIG. 15. Typical Chromatogram of an API sample, showing thecorresponding peaks for Phytic Acid and its related impurities(impurities profile) (Example 18)

The following examples illustrate the invention as disclosed herein andare not intended to limit the scope of the invention set forth in theclaims appended thereto.

EXAMPLES Example 1 Determination of IP6 in Rat Plasma Samples (SIM Mode)

The plasma sample underwent a purification and extraction of thecompound by protein precipitation with TCA in presence of a chelatingagent (EDTA). The supernatant was then diluted with trietylamine acetate(TEAA) and 20 μL was injected into UPLC®-MS system.

The ionization of phytic acid was assessed using negative electrosprayionization-mass spectrometry (ESI-MS).

Quantitative analysis was performed by mass spectrometry in the selectedion monitoring (SIM).

The compound was analyzed by gradient-elution reversed-phasechromatography using TEAA in aqueous solution and acetonitrile as mobilephase. The retention time of the analyte under the optimizedchromatographic conditions is 3.42 min (see chromatogram FIG. 1)

Example 2 Determination of IP6 in Dog Plasma Samples (SIM Mode)

The bioanalytical procedure developed in Example 1, was revalidated indog plasma samples with a full assessment of linearity, accuracy andprecision. The calibration curve was developed by injecting 20 μl ofblank plasma samples spiked with a known amount of IP6. Accuracy below10% and precision below 15% at intermediate concentrations was obtained,resulting in an excellent bioanalytical method.

Example 3 Determination of IP6 in Human Plasma Samples (SIM Mode)

The bioanalytical procedure developed in Example 1 was applied in humanplasma samples, but doubling the amounts of trichloroacetic acid (TCA),matrix and chelating agent. The retention time of the analyte under theoptimized chromatographic conditions was 4.05 min.

Example 4 Determination of IP6 in Rat Urine Samples (SIM Mode)

The bioanalytical procedure involved an extraction of the compound bydiluting the rat urine in presence of a chelating agent (EDTA) and noprecipitating agent was needed. The supernatant was then diluted withtrietylamine acetate (TEAA) and 50 μL was injected into the HPLC-MSsystem.

The ionization of Phytic acid was assessed using negative electrosprayionization-mass spectrometry (ESI-MS). Quantitative analysis wasperformed by mass spectrometry in the selected ion monitoring (SIM)mode.

The compound was analyzed by gradient-elution reversed-phasechromatography, using TEAA in aqueous solution and acetonitrile asmobile phase. The retention time of the analyte under the optimizedchromatographic conditions was ˜3.99 min (see FIG. 3 for thechromatogram)

Example 5 Determination of IP6 in Formulations (SIM Mode)

IP6 has been identified and quantified in formulations (solutions) ofthe active pharmaceutical ingredient. In this example, no simultaneousquantification of related impurities is performed. The solution mediaconsisted of water, 0.9% NaCl or other aqueous solutions as vehicle.

The determination and quantification of IP6 was developed using the sameprocedure as in Example 1, without any sample pretreatment apart fromdiluting the formulation to fit within the calibration curve range.

The validated analytical method was also used to evaluate the stabilityas well as the homogeneity of IP6 in unfiltered formulations. The methodwas proved to be lineal and specific.

Example 6 Determination of IP6 in Hepatocytes Culture Cells (SIM Mode)

The method described in Example 1 was successfully applied tohepatocytes culture cells. The study of the injection of several culturecells with a known amount of IP6 to the HPLC-MS showed a reliable methodfor this kind of matrix.

Example 7 Determination of IP6 in Pig Plasma Samples (SIM Mode)

The bioanalytical procedure developed in Example 1, was applied to pigplasma samples. The calibration curve was developed by injecting 50 μLof blank plasma samples spiked with a known amount of IP6. An accuracyand precision below 15% was obtained, resulting in an excellentbioanalytical method.

Example 8 Determination of IP6 and Related Impurities in Formulations

IP6 has been identified and quantified in formulations (solutions) ofthe active pharmaceutical ingredient. In this example, simultaneousdetermination of related impurities is performed, allowing thecalculation of the chromatographic purity of the API.

The determination and quantification of IP6 was developed usingpotassium hydroxide was used as mobile phase. Sodium hydroxide can bealternatively used and the addition of small proportions of isopropanolis strongly recommended.

The used column was a anion-exchange divinyl benzene polymer. The flowrate was maintained at 1 mL/min with a temperature of 35° C.

Better sensitivity, especially for impurities, was obtained when usingchemical or electrochemical ionic suppression.

The retention time for phytic acid was 24.6 minutes (see FIG. 4). Theidentity of IP6 is confirmed if the retention time of the main peak inthe assay sample is within ±0.5 minutes of the mean retention time ofthe peak corresponding to phytate for all injections of the assayworking standard. This technique allows the simultaneous determinationof the API together with its related impurities in a single chromatogramrun.

Example 9 Determination of ATP (Adenosine Triphosphate) in Rat PlasmaSamples (SIM Mode)

The bioanalytical procedure developed in Example 1, was applied to ratplasma samples, but changing the monitored mass in the SIM mode with themolecular weigh (M-1) of ATP. The calibration curve was developed byinjecting 50 μl of blank plasma samples spiked with a known amount ofATP. The retention time for ATP was 3.96 min. FIG. 4 shows a typicalchromatogram obtained with these samples.

Example 10 Determination of IP3 (Inositol Triphosphate) in Rat PlasmaSamples (SIM Mode)

The bioanalytical procedure developed in Example 1, was applied to ratplasma samples, but changing the monitored mass in the SIM mode with themolecular weight (M-1) of IP3. The calibration curve was developed byinjecting 50 μl of blank plasma samples spiked with a known amount ofIP3. The retention time for IP3 was 2.62 min. FIG. 5 shows a typicalchromatogram obtained with these samples.

Example 11 Determination of IP6 (Inositol Hexaphosphate) in Human, Ratand Dog Plasma and in Human and Rat Urine (MRM Mode)

The bionalytical method developed in Example 1 and used in example 1-10was transferred to the MRM mode in an UPLC®-MS/MS system. The use ofthis MRM mode resulted in an increase of the analytical sensitivity aswell as selectivity improvement.

The same extraction procedure was used. Chromatographic conditions werealso based on the same theory. The mass transition obtained aftercollision-induced dissociation and used for quantitative purpose of IP6was m/z 659.0>m/z 560.9. FIG. 6 shows a typical chromatogram obtainedwith these samples.

Example 12 Determination of IP6 in a Surrogate Matrix (Serum or PlasmaSurrogate Matrix)

The bioanalytical procedure developed in Example 11, was applied in asurrogate matrix. In a particular situation (e.g. constitutive levels ofthe analyte are expected in the blank matrix) a surrogate matrix couldbe used to prepare the calibration curve and to quantify IP6 inbiological samples. A similar matrix effect as well as the sameextraction recovery between surrogate matrix and biological matrix wasobserved resulting in an identical behavior in the UPLC®-MS/MS system.As a surrogate matrix for plasma or serum, a solution of 30 mg BSA(Bovine Serum Albumin)/mL in PBS was used. This modification gives ahigher sensitivity and avoids the use of natural biological matrices tobuild the calibration curve. FIG. 7 shows a typical chromatogramobtained with these samples.

Example 13 Determination of IP6 and Related Impurities in Formulations(SCAN Mode)

The chromatographic method developed for the determination of PhyticAcid permitted to determine some impurities presents in a Phytic Acidsolution, all the detected impurities (IP3, IP4, IP5 and m/z 779)coeluted with the peak of Phytic Acid; however, they were detected dueto their different molecular weight. IP5 was the most abundant impurity.

FIG. 8 shows a chromatogram obtained for this example.

Example 14 Determination of IP6 and Related Metabolites in RatHepatocytes (SCAN Mode)

Phytic Acid and its metabolites were detected after incubation of PhyticAcid in rat hepatocytes. The purification step involved a dilution ofthe pellets with KHB medium after precipitation with EDTA and finally adilution with TEAA. Only IP1 and IP2 were eluted with significantdifferences in the retention time relative to Phytic Acid.

FIGS. 9, 10 and 11 show typical chromatograms of Phytic Acid and itsmetabolites after incubation of Phytic Acid in rat hepatocytes.

Example 15 Determination of IP5 in Dog and Rat Plasma Samples (SIM Mode)

The bioanalytical procedure developed in Example 1, was applied in thedetermination of IP5 in dog and rat plasma.

IP5 was detected in the SIM mode using molecular weight (M-1) of IP5.The retention time for IP5 was 4.10 min.

FIG. 12 shows a typical chromatogram obtained for IP5 in rat plasma.

Example 16 Determination of Phytic Acid Metabolites in Rat PlasmaSamples (SIM Mode)

The bioanalytical procedure developed for the determination of PhyticAcid was applied to rat plasma samples in order to detect the maximumnumber of metabolites.

The metabolites were firstly detected in the SCAN mode and thenconfirmed and semi-quantified by SIM mode. Quantitative measurements canbe performed obtaining (i.e. synthesizing) the corresponding metabolitesto prepare standards. FIG. 13 shows a typical chromatogram obtained withthese samples.

Example 17 Determination of Metabolites of Phytic Acid in Dog PlasmaSamples (SIM Mode)

The bioanalytical procedure developed for the determination of PhyticAcid was applied to dog plasma samples in order to detect the maximumnumber of metabolites. The metabolites were firstly detected in the SCANmode and then confirmed and semi-quantified by SIM mode. Quantitativemeasurements can be performed obtaining (i.e. synthesizing) thecorresponding metabolites to prepare standards.

FIG. 14 shows a typical chromatogram obtained with these samples.

Example 18 Determination of Phytic Acid (Identity, Assay) and RelatedImpurities in Formulations for Quality Control of an API, a MedicalFood, a Reagent, a Food Additive, a Pharmaceutical Composition orNutraceutical

Phytic acid and related impurities have been identified and quantifiedin formulations in aqueous solution. This method allows the calculationof the assay and chromatographic purity of phytic acid.

The determination and quantification of phytic acid was carried out byIon Chromatography with post column derivatization by UV detection.

The ion-chromatography column used was a polystyrene 2% cross-linkedwith divinylbenzene polymer. The flow rate was maintained at 1 mL/min,setting a column temperature of 35° C.

Adequate sensitivity for phytic acid related compounds was obtained byusing post-column derivatization and UV detection.

The retention time for phytic acid was 48.06 minutes (see FIG. 15). Theidentity of phytic is confirmed when the retention time of the main peakin the assay sample is within ±0.5 minutes of the mean retention time ofthe peak corresponding to phytic acid for all injections of the assayworking standard.

This analytical technique allows the simultaneous determination ofphytic acid together with its related impurities (up to thirty five) ina single chromatographic run for quality control of an API, a medicalfood, a reagent, a food additive, a pharmaceutical composition ornutraceutical.

Based upon tentative assignments from comparison of the relativeretention times of the impurities formed with the literature method themajor impurities formed are identified as listed in Table 1.

TABLE 1 Tentative impurity peak assignments for degraded sample PeakRetention Time (min) RRT DL-Ins(1, 5, 6)P3 17.400 0.36 — 21.533 0.45 —22.467 0.47 DL-Ins(1, 2, 4, 6)P4 + Ins(1, 2, 3, 5)P4 23.709 0.49DL-Ins(1, 2, 3, 4)P4 + Ins(1, 3, 4, 6)P4 24.733 0.51 DL-Ins(1, 2, 4,5)P4 24.967 0.52 DL-Ins(1, 2, 4, 5)P4 27.233 0.57 DL-Ins(1, 2, 5, 6)P428.067 0.58 Ins(2, 4, 5, 6)P4 30.433 0.63 31.267 0.65 32.067 0.67 32.3330.67 DL-Ins(1, 4, 5, 6)P4 33.467 0.70 Ins(1, 2, 3, 4, 6)P5 36.100 0.75DL-Ins(1, 2, 3, 4, 5)P5 36.933 0.77 DL-Ins(1, 2, 4, 5, 6)P5 40.867 0.8541.100 0.86 Ins(1, 3, 4, 5, 6)P5 42.067 0.88 42.300 0.88 Phytic Acid48.067 1.00

The invention claimed is:
 1. A method for the direct identification andquantification of at least one phosphorus containing compound having amolecular weight of at least 200 daltons in a biological sample whereinsaid biological sample is obtained from a biological fluid selected fromthe group consisting of blood, plasma, serum, urine, saliva, lymphaticliquid, cerebrospinal fluid and combinations thereof, wherein saidbiological fluid has been treated with a precipitating agent, achelating agent, or a combination thereof, wherein said at least onephosphorus containing compound to be identified and quantified in thebiological sample is selected from: (a) a bisphosphonate orpolyphosphonate; (b) a hexametaphosphate; (c) a C3-C7 cycloalkylsubstituted compound with at least two —R groups wherein each —R groupis —OH, —OP(O)(OH)₂ or —P(O)(OH)₂, and wherein at least two —R groupsare independently selected from —P(O)(OH)₂ and —OP(O)(OH)₂ and saidC3-C7 cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, orcyclohelptyl; (d) ions or salts thereof; or, (e) a combination thereof,said method comprising: (i) preparing at least one standard sample of aphosphorus containing compound to be identified and quantified; (ii)preparing a biological sample containing the at least one phosphoruscontaining compound to be identified and quantified, wherein the processto prepare said biological sample comprises at least partially ortotally dissolving said biological sample to form a solution or a slurrycontaining the compound; (iii) introducing the at least one standardsample together or sequentially with the biological sample into a streamof a solvent system, wherein the solvent system is a polar solvent or asolvent mixture comprising at least one polar solvent and passing thesamples through at least one chromatography column containing smallparticles of resin wherein the column is essentially filled of the smallparticles in a stationary phase while maintaining the pressure of thesystem in the chromatography column between 5 and 1500 atm; (iv)identifying the retention time and/or quantifying the intensity of thesignal of the at least one phosphorus containing compound to beidentified and quantified by means of a mass spectrometry detector whenthe samples are eluted from the chromatography column; wherein in saiddirect identification and quantification of the at least one phosphoruscontaining compound in said biological sample is not radiolabeled orderivatized.
 2. The method according to claim 1, wherein the C3-C7cycloalkyl substituted compound is an inositol polyphosphate comprisingfrom 2 to 6 phosphate groups.
 3. The method according to claim 2,wherein the inositol polyphosphate is phytate.
 4. The method accordingto claim 1, wherein the biological fluid is an isolated sample obtainedfrom a human subject who is under a bioequivalence study.
 5. The methodaccording to claim 1, wherein the method is used to identify andquantify any metabolites of the at least one phosphorus containingcompound in a metabolic profile.
 6. The method according to claim 1,wherein the pH of the solvent system is between 7 and
 14. 7. The methodaccording to claim 1, wherein the mass spectrometry detector is a tandemmass spectrometer, a triple quadrupole spectrometer, or a singlequadrupole spectrometer.
 8. The method according to claim 1, wherein themass spectrometry detector operates under selected ion monitoring (SIM)mode, multiple reaction monitoring (MRM) mode, selected reactionmonitoring (SRM) mode, SCAN (positive/negative), or a combinationthereof.
 9. The method according to claim 1, wherein the standard sampleis a reference standard, an external standard, an internal standard, oran standard addition.